1. Field of the Invention
The present invention relates to an xcex1-agarase and a method for producing the same. Specifically, the present invention relates to an xcex1-agarase, which is useful for producing agarooligosaccharides with low degrees of polymerization having various physiological activities from agarose, and a method for producing the xcex1-agarase as well as use of the enzyme. The present invention also relates to a polypeptide having an xcex1-agarase activity and a gene encoding said polypeptide. Specifically, the present invention relates to an amino acid sequence of an xcex1-agarase, which is useful for producing agarooligosaccharides with low degrees of polymerization having various physiological activities from agarose, and a nucleotide sequence encoding the amino acid sequence. Furthermore, the present invention relates to a method for producing a polypeptide having an xcex1-agarase activity by genetic engineering. In addition, the present invention relates to a method for producing an agarooligosaccharide using a polypeptide having an xcex1-agarase activity.
2. Description of Related Art
Agarose is the principal constituent of agar. Agarose is a polysaccharide that has a structure in which D-galactose and 3,6-anhydro-L-galactose are alternately linked together through xcex1-1,3 bonds and xcex2-1,4 bonds. One must degrade agarose into smaller molecules in order to produce oligosacchaides from agar. For this purpose, methods in which agarose is chemically degraded and methods in which agarose is enzymatically digested are known. In a chemical degradation method, agarose can be hydrolyzed using an acid. In this case, xcex1-1,3 bonds are mainly cleaved. Two enzymes, xcex2-agarase which cleaves xcex2-1,4 bonds in agarose and xcex1-agarase which cleaves xcex1-1,3 bonds in agarose, are known to digest agarose.
Oligosaccharides obtained by cleaving agarose at xcex2-1,4 bonds are called as neoagarooligosaccharides. Neoagarooligosaccharides have D-galactose at their reducing ends and their degrees of polymerization are expressed by even numbers. On the other hand, oligosaccharides obtained by cleaving agarose at xcex1-1,3 bonds are called as agarooligosaccharides. Agarooligosaccharides have 3,6-anhydro-L-galactose at their reducing ends and their degrees of polymerization are expressed by even numbers. Recently, it was shown that agarooligosaccharides which have 3,6-anhydro-L-galactose at their reducing ends have physiological activities such as an apoptosis-inducing activity, a carcinostatic activity, various antioxidant activities, an immunoregulatory activity, an antiallergic activity, an anti-inflammatory activity and an activity of inhibiting xcex1-glycosidase (WO99/24447, Japanese Patent Application No. 11-11646). Based on the physiological activities, pharmaceutical compositions and functional foods or drinks containing the agarooligosaccharides as their active ingredients can be provided.
It is difficult to control the size of produced oligosaccharides in a method in which agarose is chemically degraded. In particular, it is quite difficult to selectively produce smaller oligosaccharides with low degrees of polymerization (e.g., T. Tokunaga et al., Bioscience and Industry, 49:734 (1991)). If xcex2-agarase is used, only neoagarooligosaccharides which do not have the above-mentioned physiological activities can be obtained because this enzyme cleaves only xcex2-1,4 bonds.
It is expected that agarooligosaccharides having physiological activities are produced by using xcex1-agarase which has an activity of cleaving xcex1-1,3 bonds. Known xcex1-agarases include enzymes produced by a marine Gram-negative bacterial strain GJ1B (Carbohydrate Research, 66:207-212 (1978); this strain is indicated as Alteromonas agarlyticus strain GJ1B in European Journal of Biochemistry, 214:599-607 (1993)) and a bacterium of genus Vibrio (JP-A 7-322878; strain JT0107-L4) However, it is impossible to produce agarobiose which has notable physiological activities by using the xcex1-agarase derived from Alteromonas agarlyticus strain GJ1B because the enzyme cannot digest hexasaccharides or shorter oligosaccharides. Furthermore, the xcex1-agarase derived from a bacterium of genus Vibrio cannot be used for the production of agarooligosaccharides using agarose as a raw material because this enzyme exhibits its activity only on hexasaccharides and shorter oligosaccharides and does not act on agarose at all.
As described above, prior art has problems regarding the production of smaller agarooligosaccharides such as agarobiose and agarotetraose which have 3,6-anhydro-L-galactose at their reducing ends and have various physiological activities.
The main object of the present invention is to provide a polypeptide having an xcex1-agarase activity which can be used for efficient production of smaller agarooligosaccharides, an amino acid sequence of the polypeptide, a gene encoding the polypeptide, a method for producing the polypeptide and a method for producing the smaller agarooligosaccharides.
In view of the problems as described above, the present inventors have studied intensively and conducted search in order to obtain an enzyme that cleaves xcex1-1,3 bonds in agarose and generates agarooligosaccharides having notable physiological activities. As a result, the present inventors have successfully found two microbial strains that produce enzymes having properties suitable for this purpose. The enzymes produced by these microorganisms were isolated and their physical and chemical as well as enzymatic properties were elucidated. Furthermore, the present inventors have successfully isolated genes for the enzymes, and found a method for readily producing polypeptides having xcex1-agarase activities by means of genetic engineering using the genes, thereby completing the present invention.
The present invention is outlined as follows. The first aspect of the present invention relates to a novel xcex1-agarase having the following physical and chemical properties:
(1) action: hydrolyzing an xcex1-1,3 bond between 3,6-anhydro-L-galactose and D-galactose;
(2) substrate specificity: acting on agarose, agarohexaose and agarooligosaccharides longer than agarohexaose but not on agarotetraose;
(3) optimal temperature: exhibiting its enzymatic activity at a temperature of 55xc2x0 C. or below; and
(4) heat stability: retaining 20% or more of its activity after treatment at 48xc2x0 C. for 30 seconds.
Such xcex1-agarases are exemplified by an enzyme that contains an amino acid sequence consisting of 749 residues from amino acid number 177 to amino acid number 925 in the amino acid sequence of SEQ ID NO:14, or an amino acid sequence in which one or more amino acids are substituted, deleted, added and/or inserted in said amino acid sequence consisting of 749 residues, or an enzyme that contains an amino acid sequence consisting of 767 residues from amino acid number 184 to amino acid number 950 in the amino acid sequence of SEQ ID NO:15, or an amino acid sequence in which one or more amino acids are substituted, deleted, added and/or inserted in said amino acid sequence consisting of 767 residues.
The second aspect of the present invention relates to a gene encoding a polypeptide having an xcex1-agarase activity, which encodes the xcex1-agarase of the first aspect. Such genes are exemplified by a gene that contains a nucleotide sequence consisting of 2247 bases from base number 529 to base number 2775 in the nucleotide sequence of SEQ ID NO:12, or a nucleotide sequence in which one or more bases are substituted, deleted, added and/or inserted in said nucleotide sequence consisting of 2247 bases, or a gene that contains a nucleotide sequence consisting of 2301 bases from base number 550 to base number 2850 in the nucleotide sequence of SEQ ID NO:13, or a nucleotide sequence in which one or more bases are substituted, deleted, added and/or inserted in said nucleotide sequence consisting of 2301 bases.
The third aspect of the present invention relates to a gene that is hybridizable to the gene of the second aspect under stringent conditions and encodes the xcex1-agarase of the first aspect.
The fourth aspect of the present invention relates to a recombinant DNA molecule that contains the gene of the second or third aspect.
The fifth aspect of the present invention relates to a transformant harboring the recombinant DNA molecule of the fourth aspect.
The sixth aspect of the present invention relates to a method for producing a polypeptide having an xcex1-agarase activity, comprising culturing a microorganism capable of producing an xcex1-agarase (e.g., a microorganism belonging to a genus to which a microorganism TKR1-7AGxcex1 (FERM BP-6990) or a microorganism TKR4-3AGxcex1 (FERM BP-6991) belongs) and collecting the xcex1-agarase of the first aspect from the culture. Both of the microorganisms TKR1-7AGxcex1 and TKR4-3AGxcex1 were deposited under Budapest Treaty on Jan. 26, 1999 (the date of the original deposit) at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan under accession numbers FERM BP-6990 and FERM BP-6991, respectively.
The seventh aspect of the present invention relates to a method for producing a polypeptide having an xcex1-agarase activity, comprising culturing the transformant of the fifth aspect and collecting the xcex1-agarase of the first aspect from the culture.
The eighth aspect of the present invention relates to a method for producing an agarooligosaccharide, comprising digesting agarose using the xcex1-agarase of the first aspect and collecting an agalooligosaccharide from the resulting digest.
The ninth aspect of the present invention relates to a novel xcex1-agarase. Such xcex1-agarases are exemplified by an enzyme containing an amino acid sequence consisting of 591 residues from amino acid number 335 to amino acid number 925 in the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence in which one or more amino acids are substituted, deleted, added and/or inserted in said amino acid sequence consisting of 591 residues, or an enzyme containing an amino acid sequence consisting of 586 residues from amino acid number 365 to amino acid number 950 in the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence in which one or more amino acids are substituted, deleted, added and/or inserted in said amino acid sequence consisting of 586 residues.
The tenth aspect of the present invention relates to a gene encoding a polypeptide having an xcex1-agarase activity, which encodes the xcex1-agarase of the ninth aspect. Such genes are exemplified by a gene containing a nucleotide sequence consisting of 1773 bases from base number 1003 to base number 2775 in the nucleotide sequence of SEQ ID NO: 12, or a nucleotide sequence in which one or more bases are substituted, deleted, added and/or inserted in said nucleotide sequence consisting of 1773 bases, or a gene containing a nucleotide sequence consisting of 1758 bases from base number 1093 to base number 2850 in the nucleotide sequence of SEQ ID NO: 13, or a nucleotide sequence in which one or more bases are substituted, deleted, added and/or inserted in said nucleotide sequence consisting of 1758 bases.
The eleventh aspect of the present invention relates to a gene that is hybridizable to the gene of the tenth aspect under stringent conditions and encodes the xcex1-agarase of the ninth aspect.
The twelfth aspect of the present invention relates to a recombinant DNA molecule that contains the gene of the tenth or the eleventh aspect.
The thirteenth aspect of the present invention relates to a transformant harboring the recombinant DNA molecule of the twelfth aspect.
The fourteenth aspect of the present invention relates to a method for producing a polypeptide having an xcex1-agarase activity, comprising culturing the transformant of the thirteenth aspect and collecting the xcex1-agarase of the ninth aspect from the culture.
The fifteenth aspect of the present invention relates to a method for producing an agarooligosaccharide, comprising digesting agarose using the xcex1-agarase of the ninth aspect and collecting an agarooligosaccharide from the resulting digest.